肺癌miR-146a表达及其对细胞增殖、侵袭及迁移的影响

目的探讨微小RNA-146a(miR-146a)在肺癌组织和细胞中的表达和甲基化状态,及其对A549细胞增殖、侵袭、迁移的影响及可能机制。方法收集2018年1月至2019年4月在我院行根治性手术的非小细胞肺癌组织和对应癌旁组织,实时荧光定量PCR(QPCR)和甲基化特异性PCR(MSP)检测miR-146a的表达水平和甲基化状态,并分析甲基化状态与肺癌临床病理特征的关系。采用5-氮杂-2’-脱氧胞苷(5-AZA-2’-dC)处理A549细胞(处理组),MTT、Transwell实验和划痕实验检测处理组细胞增殖、侵袭和迁移活性。采用Western blotting检测Notch1和发状分裂相关增强子1(Hes-1)蛋白表达。结果肺癌组织和A549细胞中miR-146a表达量分别为0.63±0.28、0.85±0.11,均低于癌旁正常组织和BEAS-2B细胞(P<0.05)。MSP检测显示肺癌组织miR-146a甲基化率为62.5%(50/80),高于癌旁组织(P<0.05);miR-146a甲基化状态与肿瘤直径、TNM分期、淋巴结转移有关(P<0.05)。处理组细胞miR-146a表达水平为2.15±0.48,高于空白对照组(P<0.05);处理组细胞增殖、侵袭和迁移活性均低于空白对照组(P<0.05)。处理组Notch1蛋白和Hes-1蛋白表达水平分别为0.24±0.05和0.22±0.06,均低于空白对照组(P<0.05)。结论启动子异常甲基化导致在肺癌组织和细胞中miR-146a表达量降低,可能通过减弱对Notch1/Hes-1信号通路的抑制作用,促进肺癌细胞增殖、侵袭和转移,miR-146a有望成为肺癌新的生物治疗靶点。     

The relationships among monocyte subsets,miRNAs and inflammatory cytokines in patients with acute myocardial infarction

Background

Acute myocardial infarction (AMI) causes irreversible myocardial damage and release of inflammatory mediators, including cytokines, chemokines and miRNAs. We aimed to investigate changes in the levels of cytokines (IL-6, TNF-α and IL-10), miRNAs profiles (miR-146 and miR-155) and distribution of different monocyte subsets (CD14++CD16-, CD14++CD16+, CD14+CD16++) in the acute and post-healing phases of AMI.

益气逐瘀方对缺氧诱导乳鼠心肌细胞凋亡及miR-24/Bim通路的影响

目的研究益气逐瘀方(参元丹)对缺氧诱导乳鼠心肌细胞凋亡及miR-24/Bim通路的影响。方法分离并培养乳鼠心肌细胞,建立心肌细胞缺氧模型,Lipofectamine2000转染miR-24 mimic/inhibitor至心肌细胞,实验分为正常组、缺氧组、参元丹组、参元丹+miR-24 mimic组、参元丹+miR-24 inhibitor组,治疗组予参元丹含药血清干预。比色法检测心肌细胞培养基上清液LDH、CPK活性,MTT法检测心肌细胞存活率,Annexin V/PI双染流式细胞术检测心肌细胞凋亡,qRT-PCR检测心肌细胞miR-24、Bim mRNA表达。结果与低氧组相比,参元丹组、参元丹+miR-24 mimic组、参元丹+miR-24 inhibitor组均能显著降低细胞培养基上清液LDH、CPK活性(P<0.05),提高心肌细胞存活率(P<0.05),降低心肌细胞凋亡(P<0.05),升高心肌细胞miR-24 mRNA表达(P<0.05),降低Bim mRNA表达(P<0.05);治疗组之间比较,参元丹+miR-24 mimic组作用更显著(P<0.05)。结论益气逐瘀方参元丹能够减轻缺氧诱导乳鼠心肌细胞损伤及细胞凋亡,其作用机制可能与其上调miR-24表达,同时抑制其靶基因Bim mRNA表达有关。     

miR-142-5p通过影响上皮间质转化抑制肺腺癌H1650细胞的侵袭与迁移

目的：探讨肺腺癌组织中miR-142-5p的表达及其对H1650细胞增殖、侵袭、迁移及上皮间质转化（epithelieal-mesenchymal transition，EMT）的影响及其作用机制。方法：收集2014年1月至2015年1月在河北医科大学第四医院胸外科行肿瘤切 除并经病理证实的107例肺腺癌患者的癌组织及其癌旁组织标本，以及人肺腺癌细胞系H1650、HCC827、 A549、 H1975、PC9和人 支气管上皮细胞BEAS-2B， 用qPCR实验检测肺腺癌组织及细胞中miR-142-5p的表达水平及其与患者临床特征的关系。分别用 miR-142-5p模拟物（mimics）、miR-阴性对照质粒（miR-NC）转染H1650细胞后， 用CCK8、细胞划痕愈合和Transwell侵袭实验分 别检测H1650细胞的增殖、侵袭和迁移能力。使用生物信息学工具预测miR-142-5p的靶基因，通过双荧光素酶报告基因实验验 证miR-142-5p对靶基因的调控作用，Western blotting检测细胞周期蛋白依赖性激酶5（cyclin-dependent kinase 5，CDK5）及EMT 相关蛋白的表达水平。结果：肺腺癌组织及细胞系中miR-142-5p表达水平显著低于癌旁组织及BEAS-2B细胞（均P<0.01）；107 例肺腺癌组织中， 61例（57.01%）低表达miR-142-5p，其表达水平与患者的TNM分期、淋巴结转移密切相关（均P<0.01）。转染 miR-142-5p模拟物后， H1650细胞中miR-142-5p高表达，细胞的增殖、侵袭和迁移能力显著降低（均P<0.05或P<0.01）。生物信息 学工具预测CDK5是miR-142-5p的靶基因，经双荧光素酶报告基因验证，miR-142-5p可显著降低H1650细胞中CDK5的表达水 平，显著提高E-cadherin表达，降低N-cadherin和Snail的表达水平（均P<0.01）。结论：miR-142-5p在肺腺癌组织和细胞中呈低表 达状态，其通过下调CDK5表达影响EMT抑制H1650细胞的侵袭与迁移能力。     

Different expression levels of exosomal miR-503 in peritoneal dialysis effluent from patients of different peritoneal transport characteristics and bioinformatics analysis

Objective To compare the expression level of exosomal miR-503 in peritoneal dialysis effluent (PDE) from patients of different peritoneal transport characteristics, predict the target genes of miR-503 and provide bioinformatic data for researches of peritoneal transport characteristics. Methods Twenty-four stable peritoneal dialysis (PD) patients were selected and divided into high transport group (H group, n=12) and low transport group (L group, n=12) according to the results of peritoneal equilibration tests (PET). The 500 ml PDE that was left on the patient's abdomen overnight was collected and concentrated using ultrafiltration cell. Exosomes in PDE were resuspended in phosphate buffered saline (PBS) after ultracentrifugation and the characteristics of PDE exosomes were identified by transmission electron microscope (TEM), nanoparticle tracking analysis (NTA), Western blotting and fluorescent staining. MicroRNAs were extracted from PDE exosomes. The expression levels of PDE exosomal miR-503 in the two groups were detected by quantitative real-time PCR. Then the relations between the relative quantity of PDE exosomal miR-503 and PET values or 24 h ultrafiltration volume (UF) were analyzed. Targetscan and miRDB databases were used to predict the target genes of miR-503. Gene ontology (GO) functional enrichment and Kyoto encyclopedia of genes and genomes (KEGG) signaling pathway analysis were relied on DAVID (https://david.ncifcrf.gov/). Results The exosomes in PDE showed a round and cup-shaped morphology under TEM, and the diameters were approximately 100 nm measured by NTA. The specific biomarkers of exosomes, CD63, CD81 and heat shock protein -70 (HSP-70) were all detected by Western blotting. The internalization and uptake of the exosomes was observed after fluorescent staining. The relative expression level of PDE exosomal miR-503 in H group was found to be significantly higher than that in L group (P=0.002), and the relative quantity of PDE exosomal miR-503 was significantly positively correlated with PET values (r=0.547, P=0.006), but not 24 h UF (r=-0.297, P=0.159). There were 156 target genes of miR-503 in total that could be predicted by two different databases at the same time. GO analysis of these 156 target genes was mainly focused on kinase binding, regulation of protein modification and catabolic process as well as regulation of epithelial cell proliferation. KEGG enriched many tumor associated or classical signaling pathways, including transforming growth factor-β (TGF-β) signaling pathway and vascular endothelial growth factor (VEGF) signaling pathway. The prediction showed that vascular endothelial growth factor A (VEGFA) was a direct target gene of miR-503 and it was also related to many proteins involved in fibrosis mechanism. Conclusions The expression level of PDE exosomal miR-503 is significantly higher in H group, and positively correlates with PET values, which may regulate the angiogenesis of peritoneal vessels by targeting VEGFA.     

外周血miR-150-5p、SOCS1 mRNA对类风湿关节炎“病”“证”诊断意义的初步探讨＊

目的 探讨类风湿关节炎患者外周血miR-150-5p、细胞因子信号抑制因子1（suppressor of cytokine signaling 1，SOCS1）mRNA的表达及对类风湿关节炎（Rheumatoid Arthritis，RA）疾病诊断、中医证型判断的意义。方法 纳入符合诊断标准的RA患者57例及健康对照组19例，根据《22个专业95个病种中医诊疗方案》有关RA的中医证候诊断标准，判断RA的中医证型。qPCR检测RA患者及健康对照组miR-150-5p、SOCS1mRNA的相对表达水平，同时检测血常规、肝功能、肾功能等常规指标。双荧光素酶分析方法判断两者是否存在靶向关系。统计分析miR-150-5p、SOCS1 mRNA对RA疾病的诊断意义及其与中医证型的相关性。结果 RA患者外周血miR-150-5p的相对表达水平下调，低于正常人群（t = -19.019，P < 0.05）；其表达水平随疾病活动度升高，有下降趋势；患者外周血SOCS1 mRNA的相对表达水平上调，低于正常人群（t = 5.333，P < 0.05）；其表达水平随疾病活动度升高，有上升趋势。MiR-150-5p与SOCS1 mRNA有靶向结合关系（P < 0.05）。通过AUC曲线比较，miR-150-5p的相对表达水平区分RA的敏感性及特异性分别为98.1%、92.1%（AUC = 0.972，P < 0.05）；SOCS1 mRNA的相对表达水平无法区分RA（AUC = 0.472，P > 0.05）。RA患者中miR-150-5p的相对表达水平低于3.06，RA患者风湿痹阻证、寒湿痹阻证的相对风险分别为8.33、250.00（P < 0.05）。结论 miR-150-5p、SOCS1 mRNA在RA患者中有差异性表达，且有靶向结合关系。miR-150-5p可能是RA的疾病诊断及中医风湿痹阻证、寒湿痹阻证证型诊断的潜在生物标志物。     

Long Noncoding RNA UCA1 Targets miR-122 to Promote Proliferation,Migration, and Invasion of Glioma Cells

Glioma is the most common and lethal malignant intracranial tumor. Long noncoding RNAs (lncRNAs) have been identified as pivotal regulators in the tumorigenesis of glioma. However, the role of lncRNA urothelial carcinoma-associated 1 (UCA1) in glioma genesis is still unknown. The purpose of this study was to investigate the underlying function of UCA1 on glioma genesis. The results demonstrated that UCA1 was upregulated in glioma tissue and indicated a poor prognosis. UCA1 knockdown induced by si-UCA1 significantly suppressed the proliferative, migrative, and invasive activities of glioma cell lines (U87 and U251). Bioinformatics analysis and luciferase reporter assay verified the complementary binding within UCA1 and miR-122 at the 3¢-UTR. Functional experiments revealed that UCA1 acted as an miR-122 “sponge” to modulate glioma cell proliferation, migration, and invasion via downregulation of miR-122. Overall, the present study demonstrated that lncRNA UCA1 acts as an endogenous sponge of miR-122 to promote glioma cell proliferation, migration, and invasion, which provides a novel insight and therapeutic target in the tumorigenesis of glioma.     

miR-155 increases stemness and decitabine resistance in triple-negative breast cancer cells by inhibiting TSPAN5

Effective therapeutic targets for triple-negative breast cancer (TNBC), a special type of breast cancer (BC) with rapid metastasis and poor prognosis, are lacking, especially for patients with chemotherapy resistance. Decitabine (DCA) is a Food and Drug Administration-approved DNA methyltransferase inhibitor that has been proven effective for the treatment of tumors. However, its antitumor effect in cancer cells is limited by multidrug resistance. Cancer stem cells (CSCs), which are thought to act as seeds during tumor formation, regulate tumorigenesis, metastasis, and drug resistance through complex signaling. Our previous study found that miR-155 is upregulated in BC, but whether and how miR-155 regulates DCA resistance is unclear. In this study, we demonstrated that miR-155 was upregulated in CD24⁻CD44⁺ BC stem cells (BCSCs). In addition, the overexpression of miR-155 increased the number of CD24⁻CD44⁺ CSCs, DCA resistance and tumor clone formation in MDA-231 and BT-549 BC cells, and knockdown of miR-155 inhibited DCA resistance and stemness in BCSCs in vitro. Moreover, miR-155 induced stemness and DCA resistance by inhibiting the direct target gene tetraspanin-5 (TSPAN5). We further confirmed that overexpression of TSPAN5 abrogated the effect of miR-155 in promoting stemness and DCA resistance in BC cells. Our data show that miR-155 increases stemness and DCA resistance in BC cells by targeting TSPAN5. These data provide a therapeutic strategy and mechanistic basis for future possible clinical applications targeting the miR-155/TSPAN5 signaling axis in the treatment of TNBC.     

MiR-92b-3p ameliorates inflammation and autophagy by targeting TRAF3 and suppressing MKK3-p38 pathway in caerulein-induced AR42J cells

Acute pancreatitis (AP) is an inflammatory disease with high morbidity and mortality. Dysregulation of microRNAs (miRNAs) was involved in human diseases, including AP. However, the effects of miR-92b-3p on AP process and its mechanism remain not been fully clarified. The expression levels of miR-92b-3p and tumor necrosis factor receptor-associated factor-3 (TRAF3) were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of TRAF3, tumor necrosis factor α (TNF-α) TNF-α, interleukin-6 (IL-6), phosphorylated mitogen-activated protein kinase kinase 3 (p-MKK3), MKK3, p38 and phosphorylated p38 (p-p38) were detected by western blot. The concentration of TNF-α and IL-6 in the medium was measured using ELISA kits. The possible binding sites of miR-92b-3p and TRAF3 were predicted by TargetScan and verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The expression level of miR-92b-3p was decreased and TRAF3 expression was increased in AR42J cells stimulated with caerulein. Moreover, the protein levels of pro-inflammatory cytokines (TNF-α and IL-6) were markedly elevated, and the expression levels of autophagy-related markers Beclin1 as well as the ratio of LC3-II/I were obviously increased in AR42J cells treated with caerulein. In addition, overexpression of miR-92b-3p or knockdown of TRAF3 significantly suppressed the release of pro-inflammatory cytokines and autophagy in caerulein-induced AR42J cells. Furthermore, TRAF3 was a direct target of miR-92b-3p and its upregulation reversed the effects of miR-92b-3p overexpression on inflammatory response and autophagy. Besides, overexpression of miR-92b-3p inhibited the activation of the MKK3-p38 pathway by affecting TRAF3 expression. In conclusion, miR-92b-3p attenuated inflammatory response and autophagy by downregulating TRAF3 and suppressing MKK3-p38 pathway in caerulein-induced AR42J cells, providing a novel avenue for treatment of AP.